Project Summary/Abstract The long-term goal of this project is to understand the mechanism of age-related meibomian gland dysfunction (MGD) and evaporative Dry Eye. Recently we have shown that mouse and human meibomian glands undergo specific age-related changes including decreased acinar cell proliferation, acinar atrophy, and altered peroxisome proliferator-activated receptor gamma (PPARg) localization from cytoplasmic-vesicluar/nuclear in young to nuclear in old mice and humans. Since PPARg is a lipid sensitive, nuclear receptor implicated in regulating adipocyte and sebocyte differentiation and lipogenesis, our findings suggest that PPARg may be involved in modulating meibomian gland differentiation during aging. Based on these findings we propose that aging of the meibomian gland may result in down-regulation of PPARg leading to decreased meibocyte differentiation and lipid synthesis, gland atrophy, and a hyposecretory MGD. Currently, there is a MAJOR GAP in knowledge regarding the role of PPARg in meibomian gland function. To test this hypothesis we have develop novel imaging and cell culture systems to assess gland volume, lipid synthesis and their regulation by PPARg. Using non-linear optical (NLO) microscopy and array tomography we have volumetrically reconstructed the mouse meibomian gland and measured total, cellular and lipid volumes in young and old glands. Preliminary studies suggest that atrophy of aging meibomian glands involves a marked loss in the lipid volume suggesting decreased meibocyte differentiation. Additionally, we have used coherent anti-stokes raman spectroscopy (CARS) to identify the regional lipid profiles within the meibomian gland and have tentatively shown that there is an age-related change in the maturation of meibomian gland lipids moving from the acini into the duct. Furthermore, we have developed an SV40 immortalized mouse meibocyte cell line that synthesizes lipids and expresses PPARg. Using these novel tools we propose the following Specific Aims. (1). Establish the age-related changes in PPARg localization and associated gene expression patterns by quantifying the subcellular localization, post-translational modification and downstream response gene expression patterns in the mouse and human meibomian gland. (2) Determine the effects of aging on the meibomian gland by quantifying the volume and lipid synthesis using NLO microscopy and array tomography to volumetrically reconstruct the meibomian gland and CARS to assess regional changes in lipid components present in the acini, ductule and duct of the mouse and human meibomian gland. (3) Assess the effects of natural and synthetic PPARg ligands on lipid synthesis by quantifying the subcellular localization, post- translational modification and downstream response gene expression patterns in cultured mouse meibocytes. (4) Measure the effect of PPARg ligands on meibocyte differentiation in vivo by quantifying the changes in PPARg expression, meibocyte proliferation, gland volume and lipid synthesis in young and old mouse meibomian glands.